Alphaviruses comprise a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. At least twenty-six known viruses and virus subtypes have been classified within the alphavirus genus, including, Sindbis virus, Semliki Forest virus (SFV), Ross River virus (RR), and Venezuelan equine encephalitis (VEE) virus.
Sindbis virus is the prototype member of the Alphavirus genus of the Togaviridae family. Its replication strategy has been well characterized in a variety of cultured cells and serves as a model for other alphaviruses. Briefly, the genome of Sindbis (like other alphaviruses) is an approximately 12 kb single-stranded positive-sense RNA molecule that is capped, polyadenylated, and contained within a virus-encoded capsid protein shell. The nucleocapsid is further surrounded by a host-derived lipid envelope, into which two viral-specific glycoproteins, E1 and E2, are inserted and anchored by a cytoplasmic tail to the nucleocapsid. Certain alphaviruses (e.g., SFV) also maintain an additional protein, E3, which is a cleavage product of the E2 precursor protein, PE2.
After virus particle adsorption to target cells, penetration, and uncoating of the nucleocapsid to release viral genomic RNA into the cytoplasm, the replicative process is mediated by the four alphaviral nonstructural proteins (nsPs) and their precursors, translated from the 5′ two-thirds of the viral genome. Synthesis of a full-length negative strand RNA, in turn, provides template for the synthesis of additional positive strand genomic RNA and an abundantly expressed 26S subgenomic RNA, initiated internally at the subgenomic junction region promoter. The alphavirus structural proteins are translated from the subgenomic 26S RNA, which represents the 3′ one-third of the genome, and like the nsPs, are processed post-translationally into the individual proteins, capsid, E1 and E2 (pE2).
Among the respiratory virus pathogens, various approaches have been disclosed for utilizing alphavirus replicon vector based vaccines as a means to stimulate immune responses against respiratory viruses such as influenza (FLU), respiratory syncytial virus (RSV) and parainfluenza virus (PIV). More specifically, among the several studies related to FLU, each have focused exclusively on the use of replicon vectors expressing a single HA or single NP antigen (see Huckriede et al., 2004, Vaccine, 22:1104-13; Berglund et al., 1999, Vaccine 17:497-507; Berglund et al., 1998, Nat. Biotechnol. 16:562-565; Pushko et al., 1997, Virology 239:389-401; Zhou et al., 1995, PNAS 92:3009-3013; Vignuzzi et al., 2001, J. Gen. Virol. 82:1737-1747; Schultz-Cherry et al., 2000, Virology 278:55-59).
The use of alphavirus based strategies that incorporate genes encoding alternative FLU antigens other than HA or NP to stimulate an immune response, or alphavirus-based immunogenic compositions or vaccines that incorporate genes encoding more than one FLU antigen, have not been described. Thus, the need exists for improved alphavirus-based FLU vaccines that address these shortcomings.
As a vaccine strategy against RSV, alphavirus vectors expressing either the G or F antigens have been examined (U.S. Pat. Nos. 6,060,308A, 6,428,324B1, and 6,475,780B1; PCT applications WO9911808 and WO 9925858; Andersson et al., 2000, FEMS Immunol. Med. Micro. 29:247-253; Fleeton et al., 2001 J. Infect. Dis. 183:1395-1398). Similarly for PIV, vectors expressing either the HN or F antigens have been suggested (U.S. Pat. Nos. 6,060,308A, 6,428,324B1, and 6,475,780B1; PCT applications WO9911808 and WO 9925858). However, the use of alphavirus based strategies that incorporate alternative RSV or PIV antigens other than HN, G, or F to stimulate an immune response, or alphavirus-based immunogenic compositions or vaccines that combine multiple RSV and PIV antigens, have not been described.
There remains a need for compositions and methods of making and using alphavirus replicon vectors, vector constructs and replicon particles as a means to more effectively stimulate an immune response to respiratory pathogens such as viruses, bacteria and fungi, and for vaccines that comprise such alphavirus-based vectors. In addition, there remains a need for compositions and methods of making and using alphavirus replicon vectors and replicon particles in effective combination or as co-expression constructs, as a means to stimulate an immune response against more than one respiratory pathogen (e.g., combination immunogen or vaccine), and for vaccines that comprise such alphavirus-based replicons.